SDS变性剂对杂色鲍碱性磷酸酶的活力与构象的影响  

Effect of sodium dodecyl sulfate (SDS) denaturant on activity and conformation of alkaline phosphatase from Haliotis diversicolor

作　　者：洪燕飞[1] 廖金花[1] 吴国欣[2] 陈清西[1] 

机构地区：[1]厦门大学生命科学学院教育部细胞生物学与肿瘤细胞工程重点实验室,福建厦门361005 [2]福建师范大学生物工程学院,福建福州350007

出　　处：《台湾海峡》2005年第2期140-144,共5页Journal of Oceanography In Taiwan Strait

基　　金：教育部跨世纪人才经费和细胞生物学与肿瘤细胞工程教育部重点实验室开放基金资助项目(2004105)

摘　　要：以十二烷基磺酸钠(SDS)为变性剂,研究其对杂色鲍碱性磷酸酶(ALP)活力的影响,结果表明酶的剩余活力随着SDS浓度增高而下降,但当下降到67%时,随着SDS浓度增高,酶活力几乎不再发生明显变化.这说明杂色鲍ALP对SDS具有高度的耐受性.研究SDS对杂色鲍ALP的pH稳定性的影响,发现该酶的pH稳定性区域由5.9～10.0区域漂移到7.6～10.6区域,显示酶的pH稳定性往碱性区域偏移.进一步以荧光和紫外光谱为监测手段,研究杂色鲍ALP经SDS微扰后的分子构象变化情况.其结果表明随着SDS浓度增高,荧光发射强度逐渐发生变化,但发射峰未发生明显变化;紫外吸收强度发生变化,但吸收峰也未发生明显变化.这说明该酶活性中心对SDS变性剂并不敏感.这在以往文献中未见过报道.The effect of sodium dodecyl sulfate (SDS) on the activity of alkaline phosphatase (ALP) from Haliotis diversicolor has been studied. The results showed that residual activity of the enzyme decreased with increasing SDS concentration.When the concentration of SDS was high to 400 mmol/dm^3, the enzyme activity just lost 33 %. After that, with increasing SDS concentrations, the activity was kept at the level of 67% of the nature enzyme, indicating the enzyme had high endurance to SDS. The effect of SDS on the pH stability of the enzyme was discussed and the results showed that the pH stability range changed from pH 5.9~10.0 to pH 7.6~10.6. The conformational changes of the enzyme in different concentrations of SDS solution were measured by fluorescence absorption spectra and ultraviolet absorption spectra. The fluorescence emission peak (at 335 nm) of the enzyme (hadn’t) shifted sharply. The fluorescence intensity decreased with increasing SDS concentrations at low concentration of SDS and rose back at high concentration of SDS. The ultraviolet absorption peak of the enzyme (hadn’t) shifted sharply, too. It was suggested that the enzyme (molecule) (hadn’t) spread sufficiently, indicating the enzyme conformation was not sensitive to SDS.

关 键 词：碱性磷酸酶 SDS 杂色鲍 变性剂 十二烷基磺酸钠 pH稳定性 浓度增高 发生变化 稳定性区域 酶活性中心 ALP 监测手段 紫外光谱 变化情况 分子构象 发射强度 吸收强度 耐受性 酶活力 吸收峰 下降 荧光 微扰 

分 类 号：O647.2[理学—物理化学] Q55[理学—化学]